Erythropoietin (EPO) is a glycoprotein hormone necessary for the maturation of erythroid progenitor cells into erythrocytes. It is produced in the kidney and is essential in regulating levels of red blood cells in the circulation. Conditions marked by low levels of tissue oxygen signal increased production of EPO, which in turn stimulates erythropoiesis. A loss of kidney function as is seen in chronic renal failure, for example, typically results in decreased production of EPO and a concomitant reduction in red blood cells.
Human urinary EPO was purified by Miyake et al. (J. Biol. Chem. 252, 5558 (1977)) from patients with aplastic anemia. However, the amount of purified EPO protein obtained from this source was insufficient for therapeutic applications. The identification and cloning of the gene encoding human EPO and expression of recombinant protein was disclosed in U.S. Pat. No. 4,703,008 to Lin. A method for purification of recombinant human erythropoietin from cell medium is disclosed in U.S. Pat. No. 4,667,016 to Lai et. al. The production of biologically active EPO from mammalian host cells has made available, for the first time, quantities of EPO suitable for therapeutic applications. In addition, knowledge of the gene sequence and the increased availability of purified protein has led to a better understanding of the mode of action of this protein.
Nephrologists often treat anemia patients with recombinant erythropoietin. Antibody-mediated pure red cell aplasia (PRCA) is a rare, but serious complication than can result from antibodies that develop in a treated patient and neutralize both the recombinant EPO and endogenous EPO. Many assays are currently in use for detection of anti-EPO antibodies, but results from different assays are currently not comparable.
The testing for anti-ESA antibodies is critical to monitor ESA safety and efficacy during clinical development and in a post-market setting [Koren E, Zuckerman L A, Mire-Sluis A R Immune responses to therapeutic proteins in humans—clinical significance, assessment and prediction. Curr Pharm Biotechnol 2002; 3(4):349-360]. A variety of analytical immunoassay methods have been described to detect and characterize anti-drug antibodies (ADAs). Each screening method offers its own unique advantages and disadvantages [Thorpe R, Swanson S J. Current methods for detecting antibodies against erythropoietin and other recombinant proteins. Clin Diagn Lab Immunol 2005; 12(1):28-39].
The most commonly used immunoassay methods in the industry for detection of binding antibodies (BAbs) are the ELISA, radioimmunoprecipitation assay (RIA), Electrochemiluminescent (ECL) assay, and Surface Plasmon Resonance Immunoassay (SPRIA), all of which have been demonstrated to detect the pathogenic antibodies in patients that develop antibody-mediated pure red cell aplasia (amPRCA) [Barger T E, Kuck A J, Chirmule N, et al. Detection of anti-ESA antibodies in human samples from PRCA and non-PRCA patients: an immunoassay platform comparison. Nephrology Dialysis Transplantation 2012; 27(2):688-693].
Although ESAs are generally well tolerated, rare cases of amPRCA have been reported [Pollock C, Johnson D W, Horl W H, et al. Pure red cell aplasia induced by erythropoiesis-stimulating agents. Clin J Am Soc Nephrol 2008; 3(1):193-199; Schellekens H, Jiskoot W. Eprex-associated pure red cell aplasia and leachates. Nat Biotechnol 2006; 24(6):613-614]. The antibody response to ESAs in patients that develop amPRCA have been previously characterized using a SPRIA and demonstrated to be a mixed IgG predominated by IgG1 and IgG4 [Mytych D T, La S, Barger T, et al. The development and validation of a sensitive, dual-flow cell, SPR-based biosensor immunoassay for the detection, semi-quantitation, and characterization of antibodies to darbepoetin alfa and epoetin alfa in human serum. J Pharm Biomed Anal 2009; 49(2):415-426; Swanson S J, Ferbas J, Mayeux P, et al. Evaluation of methods to detect and characterize antibodies against recombinant human erythropoietin. Nephron Clin Pract 2004; 96(3):c88-c95; Mytych D T, Barger T E, King C, et al. Development and characterization of a human antibody reference panel against erythropoietin suitable for the standardization of ESA immunogenicity testing. 2012]. An anti-ESA IgG1 antibody response appeared in some antibody-positive non-PRCA patients [Evens A M, Bennett C L, Luminari S. Epoetin-induced pure red-cell aplasia (PRCA): preliminary results from the research on adverse drug events and reports (RADAR) group. Best Pract Res Clin Haematol 2005; 18(3):481-489] but is also present with the detection of IgG4 in patients that develop amPRCA [Casadevall N, Nataf J, Viron B, et al. Pure Red-Cell Aplasia and Antierythropoietin Antibodies in Patients Treated with Recombinant Erythropoietin. New England Journal of Medicine 2002; 346(7):469-475]. Although the IgG1 response is considered to precede the IgG4 response, the switch is driven by the repeated and prolonged exposure to the ESA. This is also well illustrated by the analysis of antibody to grass pollen and bee venom in novice beekeepers [Aalberse R C, van der Gaag R, van Leeuwen J. Serologic aspects of IgG4 antibodies. I. Prolonged immunization results in an IgG4-restricted response. J Immunol 1983; 130(2):722-726]. The long-term administration of biological therapeutics such as adalimumab in rheumatoid arthritis (RA) patients [van Schouwenburg P A, Krieckaert C L, Nurmohamed M, et al. IgG4 Production Against Adalimumab During Long Term Treatment of RA Patients. J Clin Immunol 2012], IFN-β 1b in multiple sclerosis patients [Deisenhammer F, Reindl M, Berger T Immunoglobulin subclasses in patients with neutralizing and nonneutralizing antibodies against IFN-beta1b. J Interferon Cytokine Res 2001; 21(3):167-171] and factor VIII to hemophilia A patients [van Belden P M, van den Berg H M, Gouw S C, et al. IgG subclasses of anti-FVIII antibodies during immune tolerance induction in patients with hemophilia A. CORD Conference Proceedings 2008; 142(4):644-652] results in the development of IgG4 ADA. The development of anti-ESA IgG4 antibodies against ESAs is best studied in the nephrology patient population and has been shown to be coincident with amPRCA.
In general, serum concentrations of the IgG subclasses are not evenly distributed. The serum concentration ranges in normal adults for IgG1, IgG2, and IgG3 are 3.8 to 9.3 mg/mL, 2.4 to 7.0 mg/mL and 0.22 to 1.76 mg/mL, respectively. The total IgG4 antibody is the least abundant in serum (4% of total IgG) with a normal range of 0.04 to 0.86 mg/mL in human serum [French M M. Serum IgG subclasses in normal adults. Monogr Allergy 1986; 19:100-107]. The appearance of drug-specific IgG antibodies generally corresponds with the maturation of a secondary antibody response upon repeated exposure and generally elicits a mixed IgG subclass response [Stavnezer J J. Molecular processes that regulate class switching. Curr Top Microbiol Immunol 2000; 245(2):127-168]. The prevalence of the IgG subclasses can be antigen-specific, and the chronic exposure of a protein has been shown to develop an IgG4 isotype restriction [Shakib F F. The IgG4 subclass. Monogr Allergy 1986; 19:223-226].
In the case of the antibody response to ESAs, the greatest analytic challenge with the current immunological methods is the ability to measure the low abundance of anti-ESA specific IgG4 antibodies in the presence of much higher concentrations of the other ESA-specific IgG subclasses. The only published method to detect, but not quantitate, the anti-ESA antibody isotype is the SPRIA methodology. The challenge is that the more predominant isotypes such as IgG1 and IgG2 saturate the ESA coated surface, making it difficult to detect the less abundant anti-IgG4 antibodies.
The technology to detect specific immunoglobulins has existed for more than 50 years and has been used successfully to detect specific IgE antibodies to allergens [Maloney J M, Rudengren M, Ahlstedt S, et al. The use of serum-specific IgE measurements for the diagnosis of peanut, tree nut, and seed allergy. J Allergy Clin Immunol 2008; 122(1):145-151] and more recently the detection of antigen-specific IgG4 antibodies [Ito K, Futamura M, Moverare R, et al. The usefulness of casein-specific IgE and IgG4 antibodies in cow's milk allergic children. Clin Mol Allergy 2012; 10(1):1-1]. The advantage of this technology is the large binding capacity, allowing the quantitation of low level antigen-specific antibody isotypes such as IgG4 and IgE in a pool of other specific antibodies.
Accordingly, there is a need for anti-human EPO antibodies that can be used in sensitive, reproducible assays. Of particular need are antibodies of high- and low-affinity with binding specificity to neutralizing and non-neutralizing EPO epitopes, including IgG1, IgG2, and IgG4 subclasses of antibodies, as well as an IgM isotype. Of further need is the development of a highly sensitive and specific immunoassay for the measurement of anti-ESA IgG4 antibodies.